Jiří Macas

Repetitive DNA in the pea (Pisum sativum L.) genome: comprehensive characterization using 454 sequencing and comparison to soybean and Medicago truncatula

BackgroundExtraordinary size variation of higher plant nuclear genomes is in large part caused by differences in accumulation of repetitive DNA. This makes repetitive DNA of great interest for studying the molecular mechanisms shaping architecture and function of complex plant genomes. However, due to methodological constraints of conventional cloning and sequencing, a global description of repeat composition is available for only a very limited number of higher plants. In order to provide further data required for investigating evolutionary patterns of repeated DNA within and between species, we used a novel approach based on massive parallel sequencing which allowed a comprehensive repeat characterization in our model species, garden pea (Pisum sativum).ResultsAnalysis of 33.3 Mb sequence data resulted in quantification and partial sequence reconstruction of major repeat families occurring in the pea genome with at least thousands of copies. Our results showed that the pea genome is dominated by LTR-retrotransposons, estimated at 140,000 copies/1C. Ty3/gypsy elements are less diverse and accumulated to higher copy numbers than Ty1/copia. This is in part due to a large population of Ogre-like retrotransposons which alone make up over 20% of the genome. In addition to numerous types of mobile elements, we have discovered a set of novel satellite repeats and two additional variants of telomeric sequences. Comparative genome analysis revealed that there are only a few repeat sequences conserved between pea and soybean genomes. On the other hand, all major families of pea mobile elements are well represented in M. truncatula.ConclusionWe have demonstrated that even in a species with a relatively large genome like pea, where a single 454-sequencing run provided only 0.77% coverage, the generated sequences were sufficient to reconstruct and analyze major repeat families corresponding to a total of 35–48% of the genome. These data provide a starting point for further investigations of legume plant genomes based on their global comparative analysis and for the development of more sophisticated approaches for data mining.

RepeatExplorer: a Galaxy-based web server for genome-wide characterization of eukaryotic repetitive elements from next-generation sequence reads

MOTIVATION Repetitive DNA makes up large portions of plant and animal nuclear genomes, yet it remains the least-characterized genome component in most species studied so far. Although the recent availability of high-throughput sequencing data provides necessary resources for in-depth investigation of genomic repeats, its utility is hampered by the lack of specialized bioinformatics tools and appropriate computational resources that would enable large-scale repeat analysis to be run by biologically oriented researchers. RESULTS Here we present RepeatExplorer, a collection of software tools for characterization of repetitive elements, which is accessible via web interface. A key component of the server is the computational pipeline using a graph-based sequence clustering algorithm to facilitate de novo repeat identification without the need for reference databases of known elements. Because the algorithm uses short sequences randomly sampled from the genome as input, it is ideal for analyzing next-generation sequence reads. Additional tools are provided to aid in classification of identified repeats, investigate phylogenetic relationships of retroelements and perform comparative analysis of repeat composition between multiple species. The server allows to analyze several million sequence reads, which typically results in identification of most high and medium copy repeats in higher plant genomes.

Repeatless and Repeat-Based Centromeres in Potato: Implications for Centromere Evolution

The authors used a genome-wide approach to identify the DNA sequences associated with the centromeres of potato chromosomes. Five potato centromeres are composed of single- or low-copy sequences, including active genes; six centromeres contain almost exclusively satellite repeats. The results have implications for evolution of repeat-based centromeres from repeatless centromeres. Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats. By contrast, most newly formed centromeres (neocentromeres) do not contain satellite repeats and instead include DNA sequences representative of the genome. An unknown question in centromere evolution is how satellite repeat-based centromeres evolve from neocentromeres. We conducted a genome-wide characterization of sequences associated with CENH3 nucleosomes in potato (Solanum tuberosum). Five potato centromeres (Cen4, Cen6, Cen10, Cen11, and Cen12) consisted primarily of single- or low-copy DNA sequences. No satellite repeats were identified in these five centromeres. At least one transcribed gene was associated with CENH3 nucleosomes. Thus, these five centromeres structurally resemble neocentromeres. By contrast, six potato centromeres (Cen1, Cen2, Cen3, Cen5, Cen7, and Cen8) contained megabase-sized satellite repeat arrays that are unique to individual centromeres. The satellite repeat arrays likely span the entire functional cores of these six centromeres. At least four of the centromeric repeats were amplified from retrotransposon-related sequences and were not detected in Solanum species closely related to potato. The presence of two distinct types of centromeres, coupled with the boom-and-bust cycles of centromeric satellite repeats in Solanum species, suggests that repeat-based centromeres can rapidly evolve from neocentromeres by de novo amplification and insertion of satellite repeats in the CENH3 domains.

Selfish supernumerary chromosome reveals its origin as a mosaic of host genome and organellar sequences

Supernumerary B chromosomes are optional additions to the basic set of A chromosomes, and occur in all eukaryotic groups. They differ from the basic complement in morphology, pairing behavior, and inheritance and are not required for normal growth and development. The current view is that B chromosomes are parasitic elements comparable to selfish DNA, like transposons. In contrast to transposons, they are autonomously inherited independent of the host genome and have their own mechanisms of mitotic or meiotic drive. Although B chromosomes were first described a century ago, little is known about their origin and molecular makeup. The widely accepted view is that they are derived from fragments of A chromosomes and/or generated in response to interspecific hybridization. Through next-generation sequencing of sorted A and B chromosomes, we show that B chromosomes of rye are rich in gene-derived sequences, allowing us to trace their origin to fragments of A chromosomes, with the largest parts corresponding to rye chromosomes 3R and 7R. Compared with A chromosomes, B chromosomes were also found to accumulate large amounts of specific repeats and insertions of organellar DNA. The origin of rye B chromosomes occurred an estimated ∼1.1–1.3 Mya, overlapping in time with the onset of the genus Secale (1.7 Mya). We propose a comprehensive model of B chromosome evolution, including its origin by recombination of several A chromosomes followed by capturing of additional A-derived and organellar sequences and amplification of B-specific repeats.

Plant centromeric retrotransposons: a structural and cytogenetic perspective

BackgroundThe centromeric and pericentromeric regions of plant chromosomes are colonized by Ty3/gypsy retrotransposons, which, on the basis of their reverse transcriptase sequences, form the chromovirus CRM clade. Despite their potential importance for centromere evolution and function, they have remained poorly characterized. In this work, we aimed to carry out a comprehensive survey of CRM clade elements with an emphasis on their diversity, structure, chromosomal distribution and transcriptional activity.ResultsWe have surveyed a set of 190 CRM elements belonging to 81 different retrotransposon families, derived from 33 host species and falling into 12 plant families. The sequences at the C-terminus of their integrases were unexpectedly heterogeneous, despite the understanding that they are responsible for targeting to the centromere. This variation allowed the division of the CRM clade into the three groups A, B and C, and the members of each differed considerably with respect to their chromosomal distribution. The differences in chromosomal distribution coincided with variation in the integrase C-terminus sequences possessing a putative targeting domain (PTD). A majority of the group A elements possess the CR motif and are concentrated in the centromeric region, while members of group C have the type II chromodomain and are dispersed throughout the genome. Although representatives of the group B lack a PTD of any type, they appeared to be localized preferentially in the centromeres of tested species. All tested elements were found to be transcriptionally active.ConclusionsComprehensive analysis of the CRM clade elements showed that genuinely centromeric retrotransposons represent only a fraction of the CRM clade (group A). These centromeric retrotransposons represent an active component of centromeres of a wide range of angiosperm species, implying that they play an important role in plant centromere evolution. In addition, their transcriptional activity is consistent with the notion that the transcription of centromeric retrotransposons has a role in normal centromere function.

Comparative Genome Analysis Reveals Divergent Genome Size Evolution in a Carnivorous Plant Genus

The C‐value paradox remains incompletely resolved after >40 yr and is exemplified by 2,350‐fold variation in genome sizes of flowering plants. The carnivorous Lentibulariaceae genus Genlisea, displaying a 25‐fold range of genome sizes, is a promising subject to study mechanisms and consequences of evolutionary genome size variation. Applying genomic, phylogenetic, and cytogenetic approaches, we uncovered bidirectional genome size evolution within the genus Genlisea. The Genlisea nigrocaulis Steyerm. genome (86 Mbp) has probably shrunk by retroelement silencing and deletion‐biased double‐strand break (DSB) repair, from an ancestral size of 400 to 800 Mbp to become one of the smallest among flowering plants. The G. hispidula Stapf genome has expanded by whole‐genome duplication (WGD) and retrotransposition to 1550 Mbp. Genlisea hispidula became allotetraploid after the split from the G. nigrocaulis clade ∼29 Ma. Genlisea pygmaea A. St.‐Hil. (179 Mbp), a close relative of G. nigrocaulis, proved to be a recent (auto)tetraploid. Our analyses suggest a common ancestor of the genus Genlisea with an intermediate 1C value (400–800 Mbp) and subsequent rapid genome size evolution in opposite directions. Many abundant repeats of the larger genome are absent in the smaller, casting doubt on their functionality for the organism, while recurrent WGD seems to safeguard against the loss of essential elements in the face of genome shrinkage. We cannot identify any consistent differences in habitat or life strategy that correlate with genome size changes, raising the possibility that these changes may be selectively neutral.

Development and Characterization of Microsatellite Markers from Chromosome 1-Specific DNA Libraries of Vicia Faba

An integrated approach has been developed for targeted retrieval of microsatellite markers from selected regions of the field bean (Vicia faba L.) genome. The procedure is based on a combination of advanced physical and genetic mapping techniques and includes the following steps: 1) flow-sorting of metaphase chromosomes, 2) construction of microsatellite-enriched chromosome-specific DNA libraries, 3) isolation of polymorphic microsatellite sequences from the libraries, 4) testing chromosome specificity of the microsatellites using polymerase chain reaction with purified fractions of individual chromosome types, and 5) integration of chromosome-specific markers into a genetic map. Several strategies for isolation of microsatellite clones were tested, including direct screening of non-enriched libraries with single or mixed probes and screening of the libraries after one or two rounds of enrichment. Finally, the usefulness of this approach was demonstrated by the retrieval of novel markers from a selected portion of the largest field been chromosome (No. 1).

Next Generation Sequencing-Based Analysis of Repetitive DNA in the Model Dioceous Plant Silene latifolia

Background Silene latifolia is a dioceous plant with well distinguished X and Y chromosomes that is used as a model to study sex determination and sex chromosome evolution in plants. However, efficient utilization of this species has been hampered by the lack of large-scale sequencing resources and detailed analysis of its genome composition, especially with respect to repetitive DNA, which makes up the majority of the genome. Methodology/Principal Findings We performed low-pass 454 sequencing followed by similarity-based clustering of 454 reads in order to identify and characterize sequences of all major groups of S. latifolia repeats. Illumina sequencing data from male and female genomes were also generated and employed to quantify the genomic proportions of individual repeat families. The majority of identified repeats belonged to LTR-retrotransposons, constituting about 50% of genomic DNA, with Ty3/gypsy elements being more frequent than Ty1/copia. While there were differences between the male and female genome in the abundance of several repeat families, their overall repeat composition was highly similar. Specific localization patterns on sex chromosomes were found for several satellite repeats using in situ hybridization with probes based on k-mer frequency analysis of Illumina sequencing data. Conclusions/Significance This study provides comprehensive information about the sequence composition and abundance of repeats representing over 60% of the S. latifolia genome. The results revealed generally low divergence in repeat composition between the sex chromosomes, which is consistent with their relatively recent origin. In addition, the study generated various data resources that are available for future exploration of the S. latifolia genome.

Stretching the Rules: Monocentric Chromosomes with Multiple Centromere Domains

The centromere is a functional chromosome domain that is essential for faithful chromosome segregation during cell division and that can be reliably identified by the presence of the centromere-specific histone H3 variant CenH3. In monocentric chromosomes, the centromere is characterized by a single CenH3-containing region within a morphologically distinct primary constriction. This region usually spans up to a few Mbp composed mainly of centromere-specific satellite DNA common to all chromosomes of a given species. In holocentric chromosomes, there is no primary constriction; the centromere is composed of many CenH3 loci distributed along the entire length of a chromosome. Using correlative fluorescence light microscopy and high-resolution electron microscopy, we show that pea (Pisum sativum) chromosomes exhibit remarkably long primary constrictions that contain 3–5 explicit CenH3-containing regions, a novelty in centromere organization. In addition, we estimate that the size of the chromosome segment delimited by two outermost domains varies between 69 Mbp and 107 Mbp, several factors larger than any known centromere length. These domains are almost entirely composed of repetitive DNA sequences belonging to 13 distinct families of satellite DNA and one family of centromeric retrotransposons, all of which are unevenly distributed among pea chromosomes. We present the centromeres of Pisum as novel “meta-polycentric” functional domains. Our results demonstrate that the organization and DNA composition of functional centromere domains can be far more complex than previously thought, do not require single repetitive elements, and do not require single centromere domains in order to segregate properly. Based on these findings, we propose Pisum as a useful model for investigation of centromere architecture and the still poorly understood role of repetitive DNA in centromere evolution, determination, and function.

Flow cytogenetics and plant genome mapping.

The application of flow cytometry and sorting (flow cytogenetics) to plant chromosomes did not begin until the mid-1980s, having been delayed by difficulties in preparation of suspensions of intact chromosomes and discrimination of individual chromosome types. These problems have been overcome during the last ten years. So far, chromosome analysis and sorting has been reported in 17 species, including major legume and cereal crops. While chromosome classification by flow cytometry (flow karyotyping) may be used for quantitative detection of structural and numerical chromosome changes, chromosomes purified by flowsorting were found to be invaluable in a broad range of applications. These included physical mapping using PCR, high-resolution cytogenetic mapping using FISH and PRINS, production of recombinant DNA libraries, targeted isolation of markers, and protein analysis. A great potential is foreseen for theuse of sorted chromosomes for construction of chromosome and chromosome-arm-specific BAC libraries, targeted isolation of low-copy (genic) sequences, high-throughput physical mapping of ESTs and other DNA sequences by hybridization to DNA arrays, and global characterization of chromosomal proteins using approaches of proteomics. This paper provides a comprehensive review of the methodology and application of flow cytogenetics, and assesses its potential for plant genome analysis.